The three-dimensional structure of the vnd/NK-2 homeodomain bound to an 18 base pair DNA duplex has been determined. Six site directed mutants involving positions 52, 54, and 56 have been expressed and purified and their binding and structural properties have been measured. Position 56 is primarily responsible for modifications in the length of the recognition helix and position 54 determines the DNA consensus sequence recognized by the homeodomain. Mutation of tyrosine in position 54 to methionine decreases the affinity of the homeodomain for the consensus sequence containing as core bases CAAG by a factor of 10. Such sequence specific DNA recognition by transcription regulators continues to be the topic of primary interest in our laboratory.